Social buffering of oxidative stress and cortisol in an endemic cyprinid fish

Fish exhibit complex social behaviours that can influence their stress levels and well-being. However, little is known about the link between social interactions and stress in wild fish, especially in running water environments. While many studies have explored the stress axis in fish, most have focused on specific social contexts, leaving gaps in understanding stress responses to social changes. Our study investigated collective behaviour and stress in wild Italian riffle dace (Telestes muticellus) in a controlled experimental setup simulating a natural river system. Results reveal that group-living fish have lower cortisol and oxidative stress levels in muscle tissue compared to solitary counterparts, suggesting a calming effect of conspecific presence. Additionally, we observed upregulated expression of antioxidant enzymes in group-living fish, indicating potential benefits to antioxidant defence systems. These insights shed light on the dynamic relationship between group behaviour and stress in wild fish within running water habitats and emphasise the use of multidisciplinary approaches.

treatment, MDA levels were found to be significantly lower in the two-fish group (p > 0.05) and highly significantly lower in the six-fish group compared to fish swimming alone (Fig. 2).
Finally, advanced oxidation protein products (AOPP) levels were found to be highly significantly lower (p > 0.01) in both the two-fish and six-fish groups, with this effect observed both in the presence and absence of a waiting period relative to the single-fish condition (Fig. 3).

Expression of antioxidant enzymes
The expression of genes encoding antioxidant enzymes was analysed by qPCR to localise oxidative stress balance in the cell (Fig. 4).Our analysis revealed two distinct expression patterns of the selected genes immediately after the end of the experiment and after waiting 10 min.Most genes showed their highest expression immediately after the treatment in both two-and six-fish groups.However, after the waiting time, single fish showed a consistently higher expression than fish groups, with only sod1 being higher in two-and six-fish conditions.).The plot indicates no significant difference in MDA levels among the groups without a waiting period.Still, there was a significant increase in MDA levels in single fish compared to the two-and six-fish groups after a waiting period (N = 10 per condition and time point).The heatmap was generated to visualise the expression levels of all genes encoding antioxidant enzymes across the different treatments and time points.Each row in the heatmap represents a single gene, while each column represents a specific experimental condition (1, 2, and 6 fish) and time combination.The heatmap colour scale represents the relative expression levels, with red indicating a higher gene expression and green indicating lower expression measured by qPCR.The results show distinct patterns of gene regulation across the different groups and time points.In particular, genes such as gpx4, prdx2, prdx4, and prdx5 showed higher expression levels in the two-fish treatment than the other treatments.In contrast, after a waiting period, gpx3, gpx4, prdx4, and cat showed a significantly higher expression levels in the single-fish treatment (p < 0.05).These findings suggest that regulating antioxidant enzyme genes in response to oxidative stress may be influenced by social context and waiting periods in fish.(Additional graph-Figure S3).

Exploration of resting times during experiments
The linear regression analysis did not reveal a significant association between normalised resting times and cortisol or malondialdehyde (MDA) levels in fish.While resting behaviour appears to be linked to oxidative stress, as indicated by the positive correlation with Advanced Oxidation Protein Products (AOPP) levels (Fig. 6), it does not directly influence cortisol and MDA levels.

Discussion
Our study provides new insights into the relationship between collective behaviour and stress in wild fish exposed to running water, shedding light on the unique mechanisms involved in maintaining social behaviour and reducing the stress response of group living.In contrast to the grouped fish conditions, a single individual in the flume led to a significant upregulation of stress levels, as demonstrated by physiological and behavioural responses.Therefore, the absence of conspecifics can lead to the activation of the hypothalamic-pituitary-interrenal (HPI) axis and the formation of ROS, resulting in increased cortisol levels and oxidative damage in the cells.
Previous studies have suggested that the presence of another individual can reduce stress responses in fish when dealing with environmental stressors, hypothesising that this is due to the increased calmness provided by social interactions 23,48 .This phenomenon is commonly referred to as social buffering, which can provide individuals with the ability to cope more effectively with stressful conditions 20,23 .By reducing stress levels, social buffering may help fish maintain normal behaviour and improve their overall well-being 38 .Conspecifics have been shown to buffer the stress response and decrease cortisol levels in several fish species 24,49,50 , highlighting www.nature.com/scientificreports/ the importance of social context in stress regulation.The availability of conspecifics may provide a sense of safety and security, leading to lower stress hormone release.Furthermore, it has been shown that social isolation and lack of social support can negatively affect animals' coping abilities and stress levels 50,51 .For example, studies on social buffering in various species have indicated that being in the presence of conspecifics can reduce the physiological and behavioural responses to stress by releasing oxytocin 52,53 .In the context of fish, research has suggested that conspecific social support can improve their ability to cope with stressors such as low oxygen levels and high water temperatures 17,51 .While social buffering has traditionally been thought to reduce stress responses in fish, our data suggest that it may also activate other physiological responses to reduce stress.In fact, in the presence of conspecifics, social buffering may reduce stress by activating the gene expression of antioxidant enzymes, which can help mitigate the adverse effects of ROS production and reduce cellular damage.Therefore, it is essential to consider other physiological responses, such as antioxidant responses, that are directly involved in stress reduction.Integrating all these parameters can provide a much more accurate picture of the stress condition, detailing what is happening at the molecular, cellular and systemic levels.
When animals are exposed to environmental changes, they may experience an excess production of ROS, which can lead to oxidative stress and damage to biomolecules, cells and tissues 54 .The HPI axis may be activated in response to this stress, releasing cortisol as a stress hormone.Cortisol, in turn, can also stimulate the production of ROS and exacerbate oxidative stress 37 .Although it may appear counterintuitive, these exhibit characteristics of a classic positive feedback control mechanism capable of rapidly activating the antioxidant defence system.Activation of the antioxidant defence system can limit the presence of ROS while limiting oxidative stress damage.
Under the experimental conditions of our study, solitary fish demonstrated a limited ability to cope with the stress of swimming in running water, resulting in increased oxidative stress.Notably, activating genes encoding antioxidant enzymes in the solitary fish group occurred only as a recovery response after removing the stressor.This transcriptional activation was observed 10 min after the end of the treatment.The induced proteins encompassed both cytoplasmic (Prdx4) and mitochondrial (SOD2, Prdx5, GPx4) enzymes.The involvement of catalase (CAT) is particularly intriguing, with increased mRNA expression possibly linked to peroxisomal proliferation, a phenomenon commonly observed in animals exposed to stressogenic conditions 55 .
In contrast, the activation of the antioxidant system was more pronounced in the two-fish and six-fish groups, with the two-fish group displaying a more robust response.The two groups exhibited quantitative and qualitative differences in their stress responses.In the two-fish group, there was widespread gene activation of enzymes involved in counteracting excessive hydrogen peroxide production, such as Prdx2, Prdx5, and GPx4.Conversely, the six-fish group showed a more significant response against the increased formation of the superoxide anion, as evidenced by the enhanced activation of the sod1 and sod2 genes.Notably, the induction of superoxide dismutases (SODs) was accompanied by the induction of Prdx2 and GPx4, which fulfil complementary roles in eliminating hydrogen peroxide produced through superoxide anion dismutation in the cytoplasm and mitochondria.
A common observation in both groups was that the primary site of ROS formation appeared to be the mitochondrion, as indicated by the involvement of genes specifically expressed in this organelle (sod2, prdx5 and gpx4) 25,38,40 .Of particular significance is the upregulation of glutathione peroxidase 4 (GPx4), suggesting its vital role as a supreme ROS scavenger and an essential component of the fish's antioxidant defence system under Figure 6.The graph displays a linear regression model to investigate the relationship between AOPP (Advanced Oxidation Protein Products) and two predictors, normalised seconds of resting time and sampling (with and without "waiting time").The Type II tests Anova showed a significant effect on the normalised seconds on AOPP levels (F = 9.8954, p < 0.01), suggesting that changes in resting times are associated with changes in AOPP.However, there was no significant effect on the time point of sampling.www.nature.com/scientificreports/stressful conditions 38,41,42 .GPx4 plays a crucial role in cellular defence against oxidative stress by detoxifying lipid peroxides generated during periods of oxidative stress.The early induction of GPx4 likely serves to cope with increased lipid peroxidation in the fish subjected to the treatment, thereby maintaining cellular homeostasis and preventing damage to cell membranes and other critical cellular components 25,41 .GPx4 operates as a vigilant scavenger at the mitochondrial level, where an initial surge in ROS production often arises due to oxygen utilisation by cytochrome c oxidase, a vital component of the electron transfer chain.This chain is integral to ATP production via ATP synthase, and the heightened response of GPx4 underscores its significance as the frontline defender against oxidative stress in fish [56][57][58] .
The observed pattern in our study indicated a return to baseline gene activation levels following the treatment, suggesting effective regulation of reactive oxygen species (ROS) production.This finding implies that a dedicated recovery phase was unnecessary as the stress response was efficiently controlled.The complexity of the stress response to being in the flume was illuminated, revealing the involvement of diverse signalling pathways and cellular processes.Furthermore, the context-specific activation of specific genes in distinct cell types or tissues, influenced by various factors, highlights the multifaceted nature of the mechanisms at play.These objective findings expand our understanding of fish's intricate stress response systems, presenting new opportunities for further investigation and exploration.
Furthermore, our study highlighted the importance of analysing stress indicators directly in the muscle tissue.Due to the direct involvement in movement and ability to monitor responses to stress and oxidative stress, myocytes are among the cells that consume the most oxygen and are, consequently, more likely to produce ROS.Moreover, stress and muscle activity can influence each other bidirectionally, whereby stress can affect movement patterns and reduce swimming ability.The use of muscle tissue as a stress bioindicator has been widely studied in larger fish species, but our study provides new insights into its usefulness in smaller fish.Measuring stress levels in muscle tissue is a good alternative in small fish, where obtaining other tissues in the amount required to carry out individual assessments is impossible.Above all, cortisol levels were positively correlated with plasma levels 31,54 , and therefore, skeletal muscles supply an alternative matrix for analysing stress levels in smaller-sized fish with low amounts of blood or other available tissue.
Nevertheless, other studies have explored the role of the central nervous system (CNS) in fish stress responses, revealing the involvement of other tissues in the stress response 59 .Indeed, the different results between the various experimental groups suggest that early activation of the antioxidant system may not depend exclusively on the levels of ROS production.Furthermore, evidence suggests that in vertebrates, the relationships between oxidative stress, antioxidant defences and CNS function are equally central to responses to the physical and social environment 56 .Unfortunately, links between cognitive performance and oxidative status remain virtually unexplored by behavioural ecologists.In particular, it is not known whether the levels of oxidative stress found in wild animals may be related to behaviour and cognitive performance, processing information in an unpredictable or adverse environment.Although we did not explore individual differences in behaviour and stress response due to the limitations of tracking individual fish in our experiment, our findings suggest that behavioural observations, such as resting times, could provide valuable insights into the stress response of fish and should be considered as part of a multifaceted approach to studying stress in fish.
The lower resting times observed in the grouped fish than in solitary fish suggest that being in a group may provide a stimulating environment that encourages swimming and reduces the likelihood of stress-related behaviour such as restlessness or immobility.Especially as it highlights the potential importance of social factors in modulating stress responses in fish, resting times can reflect the activity level or the fish's willingness to swim, as fish swim typically continuously in the wild, except when they need to rest or hide 60 .For instance, Dreosti et al. 61 found that exposure to a novel environment increased the exploratory behaviour of zebrafish while decreasing their resting behaviour compared to fish in a familiar environment.Previous research shows that fish have trouble adjusting to their surroundings 5 ; therefore, we cannot exclude that this might have also impacted our results.Fish depend on sensory cues to navigate and comprehend their environment, which can cause confusion and disorientation when disrupted or unfamiliar 12 .Fish may stop swimming and freeze at the downstream grid because they feel exposed, unlike the group, which exhibits bold behaviour and swims more.It is well known that the availability of companions increases swimming performance 62 .Moreover, previous studies have shown that even visual contact with conspecifics can enhance swimming abilities in fish 63 , which further supports the importance of social interactions for fish performance and well-being.
Therefore, the reduced coping capacity of solitary fish contributes to their extended resting periods in the downstream grid, as they may struggle to acclimate to the current and maintain their position.Our observation supports that the stress levels of solitary fish did not decrease after the 10-min waiting period, implying that they may have been more stressed and less able to cope with the environment than fish in groups.Although we provided the fish with a 5-min acclimation interval before recording, it is possible that the new circumstances in an open field with no shelters and flowing water exposed to predators first scared them.This phenomenon is evident in the six-fish condition involving higher capturing times over time.Hence, based on the available literature, fish can exhibit avoidance behaviour and altered swimming patterns in response to novel or stressful environments 12,62 , which supports the possibility that the new circumstances in our study, such as an open field with no shelters and flowing water exposed to predators, may have initially scared the fish and contributed to their reduced coping capacity.While our study primarily focused on non-migrating juvenile T. muticellus, it is essential to acknowledge that the ecological context of this species is characterized by preferences for specific habitats, such as riffle and pool areas, as well as their selection of moderate water velocities 44 .
While many aspects of the complex relationship between stress and behaviour in fish remain to be fully elucidated, our findings hold significance for understanding how these animals respond to varying environmental conditions, which is particularly pertinent to species inhabiting dynamic aquatic ecosystems.These environments can subject fish to a range of stressors 43 , impacting their stress levels and coping mechanisms.Such insights www.nature.com/scientificreports/are invaluable for devising management strategies that promote the health and well-being of fish populations especially during their juvenile stages when they exhibit specific ecological behaviours.Further research could investigate the specific mechanisms underlying the effects of social buffering on oxidative stress and antioxidant defences, particularly in the context of the CNS and how it influences physiological responses in fish.Previous research has shown that isotocin, a neuropeptide involved in social behaviour, can reduce the cortisol response to stress in fish, suggesting that social buffering may involve the CNS and modulate the HPI axis response 51,61 .Thus, our work lays the foundation for exploring these intricate relationships and their ecological ramifications, such as antipredator behaviour, spatial distribution, refuge-seeking behaviour, and foraging dynamics, all characteristic features of juvenile cyprinid fish and hold substantial ecological relevance.

Conclusion
In conclusion, our study demonstrates the effectiveness of utilising a physiological marker to assess the effects of collective behaviour in fish residing in running water environments.We gain valuable insights into the multifaceted dynamics between social interactions and physiological responses by evaluating the oxidative stress response in conjunction with cortisol release.This approach is a powerful tool for comprehending the mechanisms underlying group living and promoting organismal health and fitness.Although social fish rarely experience isolation in natural or aquaculture settings, our findings shed light on the potential physiological benefits of social interactions in fish.These discoveries significantly affect conservation efforts to preserve fish populations and their natural habitats.

Ethical approval
The

Animals
Juvenile Italian riffle dace (Telestes muticellus), an endemic fish species of Northern Italy, France, and Switzerland 44 , was used to investigate the effect of water flow velocity on fish behaviour and physiology.100 fish with a size of 5 ± 0.5 cm were caught using electrofishing in Noce stream near Pinerolo, northern Italy, in May 2021 (44°56′18.52″N07°23′11.24″E)and transported to a hatchery in Porte, Italy.Fish were kept in the food-rich river-fed tanks outdoors at 15.5 °C for at least two weeks before starting experiments.After this time, they were transferred indoors to acclimate to hatchery conditions for more than 30 h in habituation tanks at 14.5 °C.Fish were transferred to indoor tanks every three days, and experiments were carried out over 9 consecutive days.Individuals from the same habituation tank were employed for both two-fish and six-fish groups to minimize the potential for disruption and stress responses in fish used for subsequent experiments.Furthermore, habituation tanks were utilised no more than twice daily, ensuring that the fish remained in familiar social environments and minimising any potential disturbances to other fish awaiting experimentation.

Experimental setup
Experiments were carried out in a portable flume with transparent Perspex walls (30 × 60 × 30 cm).A stainlesssteel wire mesh grid delimited the swimming arena with an opening 1 cm downstream and a flow straightener upstream (which also served to generate a laminar flow).Mean flow velocity was controlled by a pump inverter and by varying the height of a downstream weir.Volumetric flow, water level, and temperature were monitored with dedicated sensors (AquaTransTM AT600, Baker Hughes; BUS0025, BALLUFF; PT100 thermoresistance) connected to a data logger (DAQ USB-6002, National Instruments).Two video cameras (Sony FDR-AX43; 1920 × 1080 pixels, 50 fps) were positioned laterally and beneath the flume to track fish positions.

Experimental protocol
The experimental setup consisted of treating the fish with three consecutive water velocities: a habituation time of 5 min at a mean flow velocity of 10 cm/s, followed by 30 min of testing at increasing velocities (10, 20, and 35 cm/s, each for 10 min).Transitions between the three different flow regimes lasted 30 s.To comprehensively assess fish behaviour and stress responses under ecologically relevant conditions, we employed three consecutive water velocities, including a gradual increase, to mimic the dynamic hydrodynamic environments often encountered by fish in natural riverine ecosystems.The water level and temperature were kept constant at 15 cm and 14.5 ± 0.5 °C, respectively.Three different group sizes of 1, 2, and 6 fish were tested in a randomised order with 10 trials of the two-and six-fish groups and 20 trials with a solitary fish (Figure S1).In this study, we utilised the six-fish treatment as a reference point for physiological measurements, considering it a control situation due to its alignment with the natural group behaviour of T. muticellus observed in the wild.While physiological measurements were not conducted throughout the entire experiment, this approach allowed us to assess how alterations in group size influenced stress responses within a context that simulates the species' typical social structure and habitat conditions.From the two and six-fish treatments, two fish were sampled from each trial for testing to achieve an equal sample size between treatments.Half of the tests were immediately transferred to an anaesthetic bath.

Figure 1 .
Figure 1.The boxplot shows the cortisol levels measured in fish muscle (ng/g) tissue under different experimental conditions.Numbers over the graphs indicate the time after the end of experiments (0 and 10 min.).The plot indicates significantly low cortisol levels in the two-fish and six-fish groups compared to the single-fish condition (p < 0.05) (N = 10 per condition and time point).

Figure 2 .
Figure 2. The boxplot displays the distribution of MDA levels measured in fish muscle tissue under different experimental conditions.Numbers over the graphs indicate the time after the end of experiments (0 and 10 min.).The plot indicates no significant difference in MDA levels among the groups without a waiting period.Still, there was a significant increase in MDA levels in single fish compared to the two-and six-fish groups after a waiting period (N = 10 per condition and time point).

Figure 3 .
Figure 3.The boxplot shows the distribution of AOPP levels measured in fish muscle tissue under different experimental conditions.Numbers over the graphs indicate the time after the end of experiments (0 and 10 min.).The plot indicates significantly low AOPP levels in the two-fish and six-fish groups compared to the single-fish condition, regardless of whether a waiting period was used (p > 0.005) (N = 10 per condition and time point).

Figure 4 .
Figure 4.The heatmap was generated to visualise the expression levels of all genes encoding antioxidant enzymes across the different treatments and time points.Each row in the heatmap represents a single gene, while each column represents a specific experimental condition (1, 2, and 6 fish) and time combination.The heatmap colour scale represents the relative expression levels, with red indicating a higher gene expression and green indicating lower expression measured by qPCR.The results show distinct patterns of gene regulation across the different groups and time points.In particular, genes such as gpx4, prdx2, prdx4, and prdx5 showed higher expression levels in the two-fish treatment than the other treatments.In contrast, after a waiting period, gpx3, gpx4, prdx4, and cat showed a significantly higher expression levels in the single-fish treatment (p < 0.05).These findings suggest that regulating antioxidant enzyme genes in response to oxidative stress may be influenced by social context and waiting periods in fish.(Additional graph-FigureS3).

Figure 5 .
Figure 5. Resting times on the downstream grid: Normalised diagram by the number of total seconds spent on the grid divided by the number of fish.Times were achieved by video observations, showing standard deviation and mean. https://doi.org/10.1038/s41598-023-47926-8 https://doi.org/10.1038/s41598-023-47926-8 study was conducted following the Declaration of Helsinki and approved by the Department of Economic Development Protection of Flora and Fauna of the Metropolitan City of Turin, No. 4457 of 29 October 2020, under Italian Decree-Law No. 73.of 19 March 1948, Italian Law No. 56 of 7 April 2014, Italian Law No. 114 of 11 August 2014, Regional Law No. 23 of 29 October 2015, and Italian Legislative Decree No. 26 of 18 August 2000, and by the Director of the Provincial Office for Hunting Fishing Parks and Forests of the Province of Cuneo, No. 3014 of 26 October 2020, under Regional Law No. 37 of 29 December 2006, Provincial Council Decree No. 109 of 13 March 2007.